DE102004045186B4 - Liposomes which stimulate the intracellular penetration of active substances, their use for the preparation of topical pharmaceutical or cosmetic compositions containing these liposomes and a screening method for finding substances for stimulating intracellular penetration - Google Patents

Abstract

Use of liposomes, characterized in that they comprise, at least in part in their structure, a substance or a mixture of substances which is / are capable of intracellular penetration of at least one active substance which is present in or promotes the liposomes is stimulated, and which is selected from the group consisting of: a) a primary, secondary, tertiary or quaternary fatty monoamine comprising a single C10 to C13 fat chain; or b) a solution of quaternized plant proteins of the formula of the type R-N (R1R2R3), where R symbolizes the plant protein molecule; R1 and R2 are independently a C1-C6 hydrocarbon group and R3 is an alkyl radical having 10 to 18 carbon atoms; as a cosmetic agent that stimulates the intracellular penetration of at least one active ingredient, for producing a topical cosmetic composition; or for the manufacture of a topical pharmaceutical composition.

Description

The invention relates generally to liposomes containing a substance which favors the intracellular penetration of a substance or drug which is carried by the liposomes. More particularly, the invention relates to the use of liposomes, characterized in that they comprise, at least in part, in their structure a substance or mixture of substances capable of / having, the intracellular penetration of at least one active agent present in the liposomes or is promoted from, and which is selected from the group consisting of: a) a primary, secondary, tertiary or quaternary fatty monoamine comprising a single C10 to C13 fatty chain; or b) a solution of quaternized plant proteins of the formula RN (R ₁ R ₂ R ₃ ), where R symbolizes the vegetable protein molecule; R ₁ and R _{2 are} independently a C1-C6 hydrocarbon group, and R ₃ is an alkyl radical having 10 to 18 carbon atoms; as a cosmetic agent that stimulates the intracellular penetration of at least one drug to produce a topical cosmetic composition; or for the preparation of a topical pharmaceutical composition.

In addition, the invention relates to liposomes which, at least in part, in their structure a solution of quaternized plant proteins of the formula RN (R ₁ R ₂ R ₃ ), wherein R symbolizes the plant protein molecule; R ₁ and R _{2 are} independently a C ₁ -C 6 hydrocarbon group and R _{3 is} an alkyl radical having from 10 to 18 carbon atoms; which stimulates the intracellular penetration of at least one drug present in or carried by the liposomes.

The invention further relates to cosmetic, dermocosmetic compositions, pharmaceutical or dermopharmaceutical compositions comprising such liposomes according to claim 17 in admixture with a cosmetically acceptable carrier.

The invention further relates to a method according to claim 27 for carrying out the screening for at least one substance which is potentially capable of stimulating the intracellular penetration of a substance or drug carried by the liposomes of the present invention.

State of the art

Recently, in the document US-A-6,071,535 Described (Hayward) cationic liposomes called CATESOMES ^TM, which are very sensitive to both the pH and the ionic strength of the surrounding medium, and which are composed of Diaminfettalkylammoniumfettacylsalzen type N _n, N _{n-dimethyl-1,} n-diaminoalkyl, wherein the diamine is referred to as DDA, and wherein the alkyl portion has a number of carbon atoms n of between 2 and 8 (see column 5, lines 37 to 39), and wherein the fat-acylated portion is provided by a fatty acid comprising 10 to Has 30 carbon atoms to form a substance called ADDA (column 5, lines 41 to 48).

Such ADDA substances are reported to be commercially available under the trade name CATEMOL from Phoenix Chemical, Somerville, New Jersey, USA, reference CATEMOL 220 and 260.

The CATESOMES are formed by compounding a fatty acid of 10 to 28 carbon atoms, for example behenic acid, to form so-called A-ADDA salts, the mixture being prepared in an equimolar ratio and at a pH between 6 and 10 to produce a Salt between the quaternary amine group of the ADDA substance and the carboxyl group of the fatty acid to form (see column 5, line 66 to column 6, line 4).

This Hayward document continues to cite the document US-A-4,721,612 , which relates to conventional liposomes whose bilayers comprise a salt form of a sterol or an organic acid, such as the tris-salt form of a sterol hemusuccinate (column 5, lines 13 to 18).

In the DE 37 13 494 C2 discloses a process for preparing a dispersion of lipid globules in an aqueous phase, wherein the lipid globules are hydrocarbon chains of 8 to 30 carbon atoms and one or more hydrophilic group (s) selected from hydroxy, etheroxide, carboxyl, phosphate and amine groups, contain.

EP 1 304 160 A1 discloses liposomes or droplets of an o / w emulsion with a shell layer of polymers.

EP 1 323 415 A1 relates to a method for coating fine particles with a lipid film.

In the WO 2004/002454 A1 describe colloidal nanoaggregates comprising the carboxylate form of a camptothecin drug in combination with a cationic molecule.

WO 00/53230 discloses lamellar particles comprising a biocompatible, biodegradable polymer and carrying a cationic charge on its surface.

In the WO 98/46199 For example, multilamellar vesicles are described which have an onion-like structure and encapsulated cationic protein hydrolysates. It is expressly pointed out that these multilamellar vesicles are not liposomes.

US 2003/0073619 A1 relates to a biodegradable, non-toxic lipopolymer comprising polyethyleneimine.

With US 2003/0044407 A1 cationic liposomes are described for the complexation of antibodies.

US 5,154,854 discloses the use of lipid droplets to stabilize a dispersion wherein the lipid droplets comprise one or more hydrocarbon chains or one or more hydrophilic groups, such as e.g. As amines.

With the DE 195 05 005 C2 describes a process for the preparation of cationized vegetable protein hydrolysates and their use for the preparation of surfactants.

DE 691 16 144 T2 relates to liposomes with protein-bound amine residues.

In the DE 41 11 982 A1 discloses liposome preparations containing a mixture of one or more lecithins with 1-90% fatty acid esterified collagen hydrolyzate.

EP 0 437 479 describes liposomes made in the presence of at least one sugar and at least one protein, polypeptide and / or oligopeptide.

DE 692 07 603 T2 relates to the lipid-mediated delivery of macromolecules into the lung for the treatment of cystic fibrosis, where the lipids contain cationized proteins that can interact spontaneously with DNA.

With the DE 102 43 626 A1 discloses an active ingredient complex containing liposomes, a penetration-enhancing cosmetic raw material and a polymer.

DE 100 63 433 A1 describes the use of liposome-encapsulated photolyase and liposome-encapsulated T4 endonuclease as MMP-1 inhibiting substances in cosmetic or pharmaceutical preparations for the prevention of light-induced aging of human skin.

Norrie et al., 1982 (Analytical Biochemistry 127, 276-281) discloses cationic liposomes containing stearylamines.

US 4,897,355 refers to liposomes formed by positively charged lipids of specific structure.

Shia et al., 2001 (Biomaterials 22 (2001) 1627-1634) describes a study using small unilamellar vesicles (SUV) made from phosphatidylcholine and coated with collagen.

Summary of the invention

A principal object of the invention is, in an unexpected manner, to solve the new technical problem which consists in providing novel liposomes which allow to increase the intracellular penetration of a substance or an active agent, advantageously into the cells of the skin or the hair while at the same time limiting the cytotoxicity or the induction of cell death of these cells. Another main object of the present invention is to solve, in an unexpected way, the technical problem described in the Providing novel liposomes which not only promote the intracellular penetration of a substance or drug, advantageously into the cells of the skin or hair, but also allow for a relatively high degree of encapsulation of the substance or drug in the liposomes achieve.

Another major object of the present invention is, in an unexpected manner, to solve the technical problem which is to provide novel cosmetic or dermocosmetic, pharmaceutical or dermopharmaceutical compositions containing liposomes having excellent intracellular penetration of a substance or drug, advantageously in the cells of the skin or hair, while limiting the cytotoxicity or the induction of cell death (or apoptosis) of these cells.

Another main object of the present invention is to solve, in an unexpected manner, a new technical problem which consists in providing a new screening method for a substance which is potentially effective, the intracellular penetration of a substance or an active agent, advantageously into the Cells of the skin or hair, and preferably also to evaluate the cytotoxicity or the induction of apoptosis of these cells.

All of these technical problems are first solved simultaneously, in a safe and reliable manner that can be used in a cosmetic or pharmaceutical and industrial scale.

• a) einem primären, sekundären, tertiären oder quaternären Fettmomoamin das eine einzige C10–C13 Fettkette umfasst, vorzugsweise einem primären oder quaternären Monoamin, das eine einzige C10–C13 Fettkette umfasst;
• b) einer Lösung aus quaternisierten Pflanzenproteinen der Formel des Typs R-N(R₁R₂R₃), wobei R das Pflanzenproteinmolekül symbolisiert; R₁ und R₂ unabhängig voneinander eine C1-C6-Kohlenwasserstoffgruppe sind und R₃ ein Alkylradikal ist, das 10 bis 18 Kohlenstoffatome aufweist;

als kosmetisches Mittel, das die intrazelluläre Penetration von mindestens einem Wirkstoff stimuliert, zur Herstellung einer topischen kosmetischen Zusammensetzung; oder zur Herstellung einer topischen pharmazeutischen Zusammensetzung, wobei die Liposomen optional mindestens eine fluoreszierende Verbindung enthalten, die in Bezug auf die intrazelluläre Penetration im Wesentlichen inert ist, zum Beispiel Pentafluorbenzoylaminofluorescein, die es ermöglicht, die Penetration sichtbar zu machen.Accordingly, in a first aspect, the present invention provides the use of liposomes according to claim 1, characterized in that they comprise, at least in part, in their structure a substance or a mixture of substances capable of, or intracellularly Penetration of at least one active agent present in or carried by the liposomes and selected from the group consisting of:

• a) a primary, secondary, tertiary or quaternary fatty momoamine comprising a single C10-C13 fatty chain, preferably a primary or quaternary monoamine comprising a single C10-C13 fatty chain;
• b) a solution of quaternized plant proteins of the formula RN (R ₁ R ₂ R ₃ ), where R symbolizes the vegetable protein molecule; R ₁ and R _{2 are} independently a C ₁ -C 6 hydrocarbon group and R _{3 is} an alkyl radical having from 10 to 18 carbon atoms;

as a cosmetic agent that stimulates the intracellular penetration of at least one drug to produce a topical cosmetic composition; or for the manufacture of a topical pharmaceutical composition, wherein the liposomes optionally contain at least one fluorescent compound which is substantially inert with respect to intracellular penetration, for example pentafluorobenzoylaminofluorescein, which makes it possible to visualize the penetration.

According to a particular embodiment of the use of liposomes according to claim 1, the liposomes are characterized in that the quaternized plant protein b) is selected from the group consisting of quaternized wheat proteins, quaternized rice proteins, quaternized soy proteins. These quaternized polymers are generally commercial products, and quaternization is generally achieved by the application of tertiary amines to the chemical groups of the starting polymer.

According to another advantageous embodiment of the use of liposomes according to claim 1, the liposomes are characterized in that the primary monoamine a) is selected from the group consisting of decylamine, undecylamine, dodecylamine, tridecylamine or a mixture of primary amines, such as mixtures of copraamines, which have a hydrocarbon chain of C10 to C13.

According to a preferred embodiment of the use of liposomes according to claim 1, the liposomes contain polyethylenimine.

According to another particular embodiment of the use of liposomes according to claim 1, the liposomes in the lipid phase comprise at least one substance which modifies the membrane of liposomes, for example a polar lipid selected from the group consisting of a triglyceride, a polar phospholipid or a polar sphingolipid, alone or in a mixture.

According to another particular embodiment of the use of liposomes according to claim 1, the above-mentioned polar phospholipid is selected from phosphatidylcholine or lecithin, phosphatidylethanolamine or cephalin, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol or cardiolipin, phosphatidylinositol, alone or in a mixture.

Advantageously, the polar phospholipid is selected from a ceramide, a sphingophospholipid, a glycosphingolipid, alone or in admixture.

According to another variant of the use of liposomes according to claim 1, the liposomes comprise at least one polar lipid or a polar sphingolipid, in particular in the form of an organic acid salt of a sterol, such as the Tris salt of a sterol hemisuccinate.

According to another advantageous embodiment of the use of liposomes according to claim 1, the liposomes comprise at least one lecithin in the lipid phase extracted from a natural source selected from the group consisting of soya, rapeseed, sunflower, lupine, peanut, sesame, pumpkin ( "Courge", "marrow"), bran oil, large-size glue dotter ("caméline", "bigseed falseflax"), calendula, linen (flax) and hemp, individually or in a mixture.

According to another embodiment of the use of liposomes according to claim 1, the liposomes may also comprise a lipid phase containing cholesterol or a derivative of cholesterol, such as cholesterol hemisuccinate, as a substance that stiffens the membranes.

According to yet another advantageous embodiment of the use of liposomes according to claim 1, the liposomes in the lipid phase may comprise at least one surfactant or a surface-active substance, as a substance which liquefies the membranes of the liposomes.

According to another particular embodiment of the use of liposomes according to claim 1, the liposomes contain a fluorescent substance that is substantially inert with respect to intracellular penetration, in particular the intracellular penetration of the skin or hair, a currently preferred example of such a fluorescent substance Substance 5-pentafluorobenzoyl-aminfluorescein comprises or is composed of, in particular in a ratio of about 0.01% by weight of the composition containing the liposomes.

According to an advantageous embodiment of the use of liposomes according to claim 1, the concentration of substance (s) which stimulate / stimulate intracellular penetration is preferably between 0.05% and 25% by weight of the composition containing the liposomes 0.5% by weight and 2.5% by weight of the composition.

According to yet another advantageous embodiment of the use of liposomes according to claim 1, the above-mentioned fatty monoamine has a carbon chain with a length of C10 to C13. Advantageously, the above-mentioned fatty monoamine is a quaternized primary monoamine which is a single fatty carbon chain of from C10 to C13.

According to yet another advantageous embodiment of the use of liposomes according to claim 1, the quaternized molecule favoring the intracellular penetration of the active ingredients is selected from a solution of quaternized plant proteins, preferably from soy proteins, which are quaternized according to the formula of the type RN (R ₁ R ₂ R ₃ ), wherein R symbolizes the vegetable protein molecule which is hydrolyzed or not, hydroxyalkylated, in particular hydroxypropylated, is or is not; R ₁ and R _{2 are} independently a C 1 -C 6 hydrocarbon group, preferably methyl or ethyl, and R _{3 is} an alkyl radical having 10 to 18 carbon atoms, and more preferably 12 carbon atoms (lauryl). Particularly preferred is the quaternized vegetable protein of the formula RN (R ₁ R ₂ R ₃ ) Lauryldimoniumhydroxypropyl-hydrolyzed soybean protein or cocodimonium hydroxypropyl-hydrolyzed soybean protein.

According to another embodiment of the use of liposomes according to claim 1, the substance or active substance present in or carried by the liposomes is selected from the group consisting of an anti-free radical generator, such as vitamin E, a flavonoid, a carotenoid, vitamin C; a depigmenting substance such as catechin, hydroquinone, arbutin, phytic acid, ellagic acid, vitamin C; a degrading (slimming) substance, such as at least one xanthine, a skin or hair pigmenting substance, such as tyrosine, tryptophan, phenylanaline, a coleus extract.

According to a second aspect, the invention provides liposomes according to claim 17, characterized in that they comprise, at least partially in their structure, a substance or a mixture of substances capable of / intracellular penetration of at least one active substance which is present in or carried by the liposomes, and which is selected from a solution of quaternized plant proteins of the formula RN (R ₁ R ₂ R ₃ ), where R symbolizes the vegetable protein molecule; R ₁ and R _{2 are} independently a C ₁ -C 6 hydrocarbon group and R _{3 is} an alkyl radical having from 10 to 18 carbon atoms, the liposomes optionally containing at least one fluorescent compound that is substantially inert with respect to intracellular penetration, For example, pentafluorobenzoylaminofluorescein, which makes it possible to visualize the penetration.

According to a particular embodiment of the liposomes according to claim 17, the liposomes are characterized in that the quaternized vegetable protein is selected from the group consisting of quaternized wheat proteins, quaternized rice proteins and quaternized soy proteins. These quaternized polymers are generally commercial products, and quaternization is generally achieved by the application of tertiary amines to the chemical groups of the starting polymer.

According to a preferred embodiment of the liposomes according to claim 17, the liposomes contain polyethylenimine.

According to another particular embodiment of the liposomes according to claim 17, the liposomes in the lipid phase comprise at least one substance which modifies the membrane of liposomes, for example a polar lipid selected from the group consisting of a triglyceride, a polar phospholipid or a polar sphingolipid , individually or in a mixture.

According to another particular embodiment of the liposomes according to claim 17, the above-mentioned polar phospholipid is selected from phosphatidylcholine or lecithin, phosphatidylethanolamine or cephalin, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol or cardiolipin, phosphatidylinositol, alone or in a mixture.

Advantageously, the polar phospholipid is selected from a ceramide, a sphingophospholipid, a glycosphingolipid, alone or in admixture.

According to another variant of the liposomes according to claim 17, the liposomes comprise at least one polar lipid or a polar sphingolipid, in particular in the form of an organic acid salt of a sterol, such as the Tris salt of a sterol hemisuccinate.

According to another advantageous embodiment of the liposomes according to claim 17, the liposomes comprise at least one lecithin in the lipid phase extracted from a natural source selected from the group consisting of. Soya, rapeseed, sunflower, lupine, peanut, sesame, pumpkin ("marrow"), bran oil, large-size glue dotter ("bigseed falseflax"), marigold, linen (flax) and hemp, single or in a mixture.

According to another embodiment of the liposomes according to claim 17, the liposomes may also comprise a lipid phase containing cholesterol or a derivative of cholesterol, such as cholesterol hemisuccinate, as a substance which stiffens the membranes.

According to yet another advantageous embodiment of the liposomes according to claim 17, the liposomes in the lipid phase may comprise at least one surfactant or a surface-active substance, as a substance which liquefies the membranes of the liposomes.

According to another particular embodiment of the liposomes according to claim 17, the liposomes contain a fluorescent substance which is substantially inert with respect to the intracellular penetration, in particular the intracellular penetration of the skin or the hair, a currently preferred example of such a fluorescent substance Pentafluorobenzoylamine fluorescein, or in particular, in a ratio of about 0.01% by weight of the composition containing the liposomes.

According to an advantageous embodiment of the liposomes according to claim 17, the concentration of substance (s) which stimulate / stimulate intracellular penetration is between 0.05% and 25% by weight of the composition containing the liposomes, preferably between 0.5% and 2.5% by weight of the composition.

According to yet another advantageous embodiment of the liposomes according to claim 17, the quaternized molecule which promotes the intracellular penetration of the active ingredients is selected from a solution of quaternized plant proteins, preferably from soy proteins, which are quaternized according to the formula RN (R ₁ R ₂ R ₃ ), wherein R symbolizes the plant protein molecule which is hydrolyzed or not, hydroxyalkylated, in particular hydroxypropylated, is or is not; R ₁ and R _{2 are} independently a C 1 -C 6 hydrocarbon group, preferably methyl or ethyl, and R _{3 is} an alkyl radical having 10 to 18 carbon atoms, and more preferably 12 carbon atoms (lauryl). Particularly preferred is the quaternized vegetable protein of the formula RN (R ₁ R ₂ R ₃ ) Lauryldimoniumhydroxypropyl-hydrolyzed soybean protein or cocodimonium hydroxypropyl-hydrolyzed soybean protein.

According to another embodiment of the liposomes according to claim 17, the substance or active substance present in or carried by the liposomes is selected from the group consisting of an anti-free radical generator such as vitamin E, a flavonoid, a carotenoid , Vitamin C or their derivatives; a depigmenting substance such as catechin, hydroquinone, arbutin, phytic acid, ellagic acid, vitamin C or their derivatives; a degrading (slimming) substance, such as at least one xanthine, a skin or hair pigmenting substance, such as tyrosine, tryptophan, phenylanaline, a coleus extract or their derivatives.

In a further aspect, the invention also relates to cosmetic or dermocosmetic compositions, pharmaceutical or dermopharmaceutical compositions comprising those liposomes according to any one of claims 17 to 23 of the present invention comprising at least one substance which stimulates intracellular penetration, optionally in a cosmetic, dermocosmetic, pharmaceutically or dermopharmaceutically acceptable carrier.

Such supports are well known to those skilled in the art and some of them are cited in the two prior art documents which have been given in the description of the prior art.

Other carriers will be apparent from the examples of the cosmetic, dermocosmetic, pharmaceutical or dermopharmaceutical compositions of the following description, which is merely illustrative and which is not intended to limit the scope of the invention in any way.

According to a non-claimed aspect there is disclosed a cosmetic care method comprising topical application of a composition to the skin or hair containing liposomes as described above or as will be apparent from the following description, which is made with reference to the examples takes place, which constitute an integral part of the invention.

According to a non-claimed aspect, there is also disclosed a method of therapeutic treatment characterized in that it comprises topical application of a skin or hair-based formulation containing liposomes as described above or resulting from the examples , being an integral part of the invention, in an amount sufficient to achieve the sought-after therapeutic treatment. In general, the liposomes comprise at least one active agent which is present in or carried by the liposomes, having an effect associated with the sought-after therapeutic treatment.

In the context of cosmetic care, there is disclosed the performance of a cosmetic care selected from the group consisting of anti-wrinkle care, anti-oxidant care, and detoxifying (slimming) care ("amincissante"; Slimming care "), a skin bleaching care (" blanchissante de la peau "," skin paling care "), a skin or hair pigmenting care.

In connection with a therapeutic treatment, the performance of a suitable therapeutic treatment of the skin or hair, depending on the pathology to be treated (symptoms) is disclosed.

The invention also relates to the use of liposomes according to the present invention as cosmetic substances, in particular for the preparation of a cosmetic composition, in particular for an anti-wrinkle effect, an anti-oxidant effect, a degrading (slimming) effect ("activité amincissante "Slimming activity"), a skin bleaching effect ("activité blanchissant de la peau";"skin paling activity "), a skin or hair pigmenting action or for the preparation of a pharmaceutical composition for each of these cases, which are preferably administered topically.

• a) einem primären, sekundären, tertiären oder quaternären Fettmonoamin, das eine einzige C10 bis C13-Fettkette umfasst; oder
• b) einer Lösung aus quaternisierten Pflanzenproteinen der Formel des Typs R-N(R₁R₂R₃), wobei R das Pflanzenproteinmolekül symbolisiert; R₁ und R₂ unabhängig voneinander eine C1-C6-Kohlenwasserstoffgruppe sind und R₃ ein Alkylradikal ist, das 10 bis 18 Kohlenstoffatome aufweist;

die potentiell in der Lage ist, die intrazelluläre Penetration einer Substanz oder eines Wirkstoffs, die/der durch die Liposomen gemäß der vorliegenden Erfindung, insbesondere Liposomen, die mindestens zum Teil in ihrer Struktur eine Substanz oder eine Mischung aus Substanzen umfassen, die ausgewählt ist/sind aus der Gruppe, bestehend aus: a) einem primären, sekundären, tertiären oder quaternären Fettmonoamin, das eine einzige C10 bis C13-Fettkette umfasst; oder b) einer Lösung aus quaternisierten Pflanzenproteinen der Formel des Typs R-N(R₁R₂R₃), wobei R das Pflanzenproteinmolekül symbolisiert; R₁ und R₂ unabhängig voneinander eine C1-C6-Kohlenwasserstoffgruppe sind und R₃ ein Alkylradikal ist, das 10 bis 18 Kohlenstoffatome aufweist,
befördert wird, zu stimulieren,
dadurch gekennzeichnet, dass es umfasst:

• a) Zubereiten einer hydro-lipidischen Mischung, welche in der Lage ist, die die Liposomen der vorliegenden Erfindung, insbesondere Liposomen, die mindestens zum Teil in ihrer Struktur eine Substanz oder eine Mischung aus Substanzen umfassen, die ausgewählt ist/sind aus der Gruppe, bestehend aus: a) einem primären, sekundären, tertiären oder quaternären Fettmonoamin, das eine einzige C10 bis C13-Fettkette umfasst; oder b) einer Lösung aus quaternisierten Pflanzenproteinen der Formel des Typs R-N(R₁R₂R₃), wobei R das Pflanzenproteinmolekül symbolisiert; R₁ und R₂ unabhängig voneinander eine C1-C6-Kohlenwasserstoffgruppe sind und R₃ ein Alkylradikal ist, das 10 bis 18 Kohlenstoffatome aufweist, zu bilden;
• b) Hinzugeben einer fluoreszierenden Verbindung, welche vorzugsweise in Bezug auf die intrazelluläre Penetration im Wesentlichen inert ist, in diese hydro-lipidische Mischung vor dem Dispersionsprozess d);
• c) Hinzugeben einer Testsubstanz, die aus der Substanz, die potentiell wirksam und in der Lage ist, die intrazelluläre Penetration zu stimulieren, ausgewählt ist und einer Referenzsubstanz in die hydro-lipische Mischung, vor oder während des Dispersionsprozesses d);
• d) Dispersionsprozess zur Zubereitung der Liposomen gemäß einem beliebigen geeigneten Verfahren aus der hydro-lipidischen Mischung, wie sie entweder oben in Schritt b) oder in Schritt c) erhalten wurde;
• e) 2 bis 48 Stunden Inkubation der Zubereitung mit Fibroblasten;
• f) Detektieren von mindestens der intrazellulären Penetration der fluoreszierenden Verbindung durch Messung der intrazellulären Fluoreszenz.

According to a further aspect, the present invention also relates to a method for carrying out the screening for at least one substance selected from

• a) a primary, secondary, tertiary or quaternary fatty monoamine comprising a single C10 to C13 fatty chain; or
• b) a solution of quaternized plant proteins of the formula RN (R ₁ R ₂ R ₃ ), where R symbolizes the vegetable protein molecule; R ₁ and R _{2 are} independently a C ₁ -C 6 hydrocarbon group and R _{3 is} an alkyl radical having from 10 to 18 carbon atoms;

which is potentially capable of intracellular penetration of a substance or active substance comprising the liposomes according to the present invention, in particular liposomes comprising, at least in part, in its structure a substance or a mixture of substances selected from are selected from the group consisting of: a) a primary, secondary, tertiary or quaternary fatty monoamine comprising a single C10 to C13 fatty chain; or b) a solution of quaternized plant proteins of the formula RN (R ₁ R ₂ R ₃ ), where R symbolizes the vegetable protein molecule; R ₁ and R _{2 are} independently a C ₁ -C 6 hydrocarbon group and R _{3 is} an alkyl radical having from 10 to 18 carbon atoms,
is promoted to stimulate
characterized in that it comprises:

• a) preparing a hydro-lipidic mixture which is capable of comprising the liposomes of the present invention, in particular liposomes which comprise, at least partly in their structure, a substance or a mixture of substances selected from the group consisting of consisting of: a) a primary, secondary, tertiary or quaternary fatty monoamine comprising a single C10 to C13 fatty chain; or b) a solution of quaternized plant proteins of the formula RN (R ₁ R ₂ R ₃ ), where R symbolizes the vegetable protein molecule; R ₁ and R _{2 are} independently a C ₁ -C 6 hydrocarbon group and R _{3 is} an alkyl radical having 10 to 18 carbon atoms;
• b) adding a fluorescent compound, which is preferably substantially inert with respect to intracellular penetration, into said hydro-lipidic mixture prior to the dispersion process d);
• c) adding a test substance selected from the substance which is potentially effective and capable of stimulating intracellular penetration and a reference substance in the hydro-lipid mixture, before or during the dispersion process d);
• d) dispersion process for preparing the liposomes according to any suitable method from the hydro-lipidic mixture as obtained either in step b) above or in step c);
• e) incubation of the preparation with fibroblasts for 2 to 48 hours;
• f) detecting at least the intracellular penetration of the fluorescent compound by measuring intracellular fluorescence.

According to another embodiment of the method for performing the screening, the result of the intracellular penetration of the fluorescent compound is compared with the intracellular penetration achieved with a reference substance, in particular polyethyleneimine.

According to yet another embodiment of the method for performing the screening, the cytotoxicity and / or the induction of apoptosis of the potentially active substance is measured, in particular on fibroblasts, preferably normal human fibroblasts, in culture.

In yet another embodiment of the method of performing the screening, the fluorescent compound, which is substantially inert with respect to intracellular penetration, is a fluorescent compound that does not spontaneously penetrate into the fibroblasts in culture.

In yet another embodiment of the method of performing the screening, the fluorescent compound comprises pentafluorobenzoylaminofluorescein or is pentafluoraminofluorescein which is used at a concentration of 0.01% by weight of the final composition containing the liposomes used to evaluate intracellular penetration ,

According to another advantageous embodiment of the method for carrying out the screening, the fluorescent compound is added in the presence of polyethyleneimine, wherein the fluorescent compound and the polyethyleneimine are present in a non-cytotoxic concentration.

The invention further relates, in a further aspect, to the use of liposomes as described above or as they result from the examples which constitute an integral part of the invention, as a cosmetic substance or for the preparation of a cosmetic composition, and in particular for an anti-wrinkle effect, an anti-oxidant effect, a slimming activity ("activité amincissante", "slimming activity"), a skin bleaching effect ("activité blanchissante de la peau";"), A skin or hair pigmenting effect.

In connection with any of the foregoing aspects, surface active agents may advantageously be added to the hydro-lipidic mixture to liquefy the membranes of the liposomes.

The present invention required the selection of a tracer which does not spontaneously penetrate, for example into the fibroblasts in culture, and which, even or after encapsulation in a liposome, causes a very low cytotoxicity, which is incorporated into the liposomes (for example, based on soybean lecithin (approximately 80%) with a high yield and which is stable after encapsulation (no release and no change in fluorescence) and which does not induce instability of the liposomes. The selected indicator is advantageously 5-pentafluorobenzoylaminofluorescein or PFB-F at a concentration of 0.01%, ie 185 μM final concentration.

Advantageously, the dispersion methods for preparing the liposomes are preferably selected from shearing methods, ultrasound, extrusion, hydration / dehydration (lyophilization), freezing / thawing, reverse phase evaporation.

The presence of the liposome (lamellar structure) is confirmed by transmission electron microscopy. The size of the liposomes is evaluated by transmission electron microscopy and laser particle size analysis (granulometry).

Advantageously, the preparation of the liposomes at a pH of 4 to 8, preferably at pH 6, made.

Advantageously, the observed intracellular penetration of an active ingredient occurs in cells of the skin.

Advantageously, the penetration of a drug into the cells of the skin is primarily targeted to fibroblasts, keratinocytes and melanocytes.

The means for detecting the intracellular penetration are selected from methods for quantification and visualization, and are preferably the quantification of the intensity of the fluorescence by spectrofluorometry and the visualization by means of optical epifluorescence microscopy ("visualization en microscopie optique à épifluorescence").

Advantageously, the molecules which, firstly, achieve stimulation of the intracellular penetration of the fluorescent indicator, are more than 10% greater than those of the negative control, containing no cationic molecule, and preferably equivalent or larger than the positive control, which is a cationic one Reference molecule, polyethyleneimine, which is used at 50 uM contains. Second, the selected molecules preferably are not expected to induce cytotoxic and / or apoptotic phenomena or to induce cytotoxic and / or apoptotic phenomena that are less than those of the cationic reference molecule (polyethylenimine used at 50 μM).

• a) einem primären oder quaternären Amin variabler Kohlenstoffkettenlänge, und vorteilhafterweise Decylamin, Undecylamin, Dodecylamin, Tridecylamin, o, oder einer Mischung aus primären Aminen, die eine Kohlenwasserstoffkette von C10 bis C13 aufweisen
• b) quaternärisierten Pflanzenproteinen, insbesondere quaternärisierten Weizenproteinen, quaternärisierten Reisproteinen, oder quaternärisierten Sojaproteinen

Vorteilhafterweise liegen die Konzentrationen der verwendeten kationischen Substanzen von 0,05% bis 25%, bevorzugt von 0,5 bis 2,5%, bevorzugt bei 1%.Advantageously, the effective molecules selected to stimulate intracellular penetration are selected from the group consisting of:

• a) a primary or quaternary amine of variable carbon chain length, and advantageously decylamine, undecylamine, dodecylamine, tridecylamine, o, or a mixture of primary amines having a hydrocarbon chain of C10 to C13
• b) quaternized vegetable proteins, especially quaternized wheat proteins, quaternized rice proteins, or quaternized soy proteins

Advantageously, the concentrations of the cationic substances used are from 0.05% to 25%, preferably from 0.5 to 2.5%, preferably 1%.

Advantageously, the stimulation of intracellular penetration is optimal for carbon chains of medium length (C10 to C13).

Advantageously, the stimulation of the intracellular penetration is optimal for a primary amine (for example, C10-NH _2), compared with a secondary amine which, for example, comprises the same two carbon chains (C10-NH-C10), steric due to the induced disability.

Advantageously, certain quaternized polymers make it possible to achieve a better stimulation factor of intracellular penetration, solely by the character of the chosen polymer itself, e.g. For example, lauryldimonium hydroxypropyl from hydrolyzed wheat protein is about six times less efficient than lauryldimonium hydroxypropyl from hydrolyzed soy protein.

Advantageously, the selected cationic molecules are less cytotoxic and / or induce less apoptosis than the reference molecule used, polyethyleneimine used at 50 μM. Advantageously, the quantification of cytotoxicity is performed by a cell viability assay which evaluates the alkaline phosphatase activity of the cell and an interleukin 1 alpha determination. Apoptosis is evaluated by the quantification of caspase-1.

Advantageously, the liposomes prepared according to the inventive method described above are used to induce an increase in the intracellular penetration of cosmetic, dermocosmetic or pharmaceutical agents used in place of the fluorescent indicator.

Other objects, features and advantages of the invention will become apparent to those skilled in the art upon reading the explanatory description which proceeds with reference to the following examples.

The examples constitute an integral part of the present invention, and any feature that appears novel in relation to the prior art from the description in its entirety, including the examples, constitutes an integral part of the invention in its function and generality ,

Therefore, each example is of general importance.

Further, in the examples, all percentages are by weight unless otherwise specified, the temperature is expressed in degrees Celsius unless otherwise specified, and the pressure is air pressure unless otherwise specified.

Description of the figures

The attached 1 to 4 show the visualization of the intracellular penetration of two preselected indicators according to the invention, PBF-F and TRITC, respectively:

1 represents fibroblasts incubated for 24 hours with liposomes containing 0.01% PBF-F with a 10X magnification achieved by the objective.

2 represents fibroblasts incubated for 24 hours with liposomes containing 0.01% PBF-F with a 40X magnification achieved by the objective.

3 represents fibroblasts incubated for 24 hours with liposomes containing 0.01% TRITC with a 100X magnification achieved by the objective.

4 represents fibroblasts incubated for 24 hours with liposomes containing 0.01% TRITC and 3% PH with a 100X magnification achieved by the objective.

Example 1: Preparation and Purification of a Liposome Encapsulating a Fluorescent Indicator

Fluorescent indicators not only have the advantage of being safe, for example, in terms of radioactivity, but are also very easy to use when it comes to quantifying and demonstrating a "visual" aspect of intracellular penetration in cell culture models.

Selection of the indicator requires preparation of liposomes containing 0.01% of various water-soluble or fat-soluble indicators and their purification to remove the unincorporated indicators prior to their application to normal human fibroblasts. A liposome control without fluorescent indicator is performed in parallel.

a) Selection of the liposome preparation protocol

Liposomes were prepared at a concentration of 20% soy lecithin dissolved in Trizma dilution buffer (Sigma, France) 55 mM, adjusted to pH 5 with 27 mM NaCl. After a magnetic stirring for 30 minutes at room temperature, the mixture was very vigorously homogenized for 10 minutes.

The liposomes were then transfected into DF medium [DMEM (Dulbecco's Modified Eagle's Medium) / Ham F12 Glutamax 50/50 volume / volume supplemented with 10% calf serum with penecellin at a final concentration of 100 UI / ml with gentamicin at a final concentration of 1 microgram / milliliter, diluted with amphotericin B at a final concentration of 1 microgram / milliliter] to achieve various soybean lecithin concentrations (0.5-1-2-3-4-5-7.5-10%).

250 μl of the liposomal suspension was added to cultures of normal human fibroblasts extracted from abdominal plastic ("abdominal plasy") and cultured in a 96-well plate. Each concentration was tested in four different wells. Cell viability was evaluated by a test with paranitrophenyl phosphate, which determines intracellular alkaline phosphatase activity, after rinsing three times with phosphate buffer pH 7.4. The percentage of living cells was calculated in terms of a control performed without the addition of liposome into the culture world (n = 6).

The results obtained show that cell viability of more than 85% was achieved with up to 7.5% lecithin. At 10% lecithin viability decreased below the acceptable limit of 75% viability.

The concentration of 5%, which allowed cell viability greater than 90% to be achieved, was selected.

b) Preparation of 5% soy lecithin liposome with a built-in fluorescent indicator

0.5g of soy lecithin, 0.01% fluorescent indicator, which was pre-solubilized according to the supplier's recommendations, was placed in a bowl and diluted with 10 ml of Trizma buffer. After a magnetic stirring for 30 minutes at room temperature, the mixture was vigorously homogenized for 10 minutes to give a liposomal solution in which the liposomes had an average size that could vary between 100 and 800 nanometers, according to the exact conditions of homogenization ,

The goal of the purification step was to separate the unencapsulated indicator fraction from the fraction on indicators encapsulated in the liposomes. For this purpose, the liposomal solution was centrifuged in a conical tube for 10 minutes with 1790 g at room temperature. The deposits (pellets) were recovered and then dissolved in 10 ml of culture medium.

The liposome negative control was performed according to the protocol described above without fluorescent indicator.

The samples were stored for 24 hours at 4 ° C in the dark. Twenty-six series of liposomes have been produced, with different types of fluorescent indicators, pH-sensitive, calcium-sensitive or other indicators.

Example 2: Selection of a fluorescent indicator which can be encapsulated in a liposome and which does not spontaneously penetrate into normal human fibroblasts in culture

The selection of the indicator required the preparation and purification of liposomes according to the protocol described in Example 1, these liposomes containing different concentrations of the various water-soluble or fat-soluble indicators (Table 1, Appendix 1). Controls - a free fluorescent indicator - were prepared by dissolving a fluorescent indicator in the dilution buffer performed at the same concentration so as to quantify its cytotoxicity and its spontaneous penetration into the fibroblasts.

The comparison with a liposome which did not contain a fluorescent indicator was performed in parallel as a negative control.

After extraction from a biopsy resulting from an abdominal plastic surgery, the fibroblasts were conveniently incubated in DF medium, i. h.: DMEM (Dulbecco's Modified Eagle's Medium) / Ham F12 Glutamax 50/50 volume / volume supplemented with 10% calf serum, with penecellin at a final concentration of 100 UI / ml, with gentamicin at a final concentration of 1 microgram / mL, with Amphotericin B at a final concentration of 1 ug / milliliter, amplified.

After centrifugation, the purified liposomes were first taken up in DF culture medium, homogenized with a vortex and then added in an amount of 1 ml per well to the fibroblasts cultured in 24 well plates (Costar MW24). The controls were made under the same conditions. An untreated control was also performed.

The fibroblasts were incubated for 24 hours at 37 ° C in the presence of the liposomes and then rinsed three times in pH 7.4 phosphate buffer. First, the fluorescence dry ( "sec évaluée á";"Evaluateddry") was evaluated by spectrofluorimetry with a Cytofluor 4000 ^® (Millipore). Secondly, the intracellular fluorescence of the molecules of interest using an Olympus ^® reverse microscope (IX70) was observed using the appropriate excitation filter (objective x10, x40 and x100). Finally, the cytotoxicity of the fluorescent indicators in a 96-well plate was evaluated by incubation for 24 hours as described in Example 1.

The results obtained show that of the 25 molecules tested, only pentafluorobenzoylaminofluorescein PFB-F (P12925 Molecular Probes) and tetramethylrhodamine isothiocyanate TRITC (T-490 Molecular Probes) after incorporation into a liposome, application to the fibroblasts for 24 hours and quantification of the Penetration of the liposomal content, gave interesting positive results.

The adjacent 1 to 4 Photographs showing the intracellular penetration of the two pre-selected indicators PFB-F and TRITC at the concentration of 0.01% in the liposomes after incubation with the fibroblasts for 24 hours visualize. Table 2: Results of indicator screening Cytotoxicity (% of untreated control) Penetration-free sample Penetration (fluorescence after washing) PFB-F (485-530) 110.7% 8 (Yield 45) 65 (Yield 45) TRITC (530-620) 74.8 295 (Yield 50) 1119 (Yield 50)

Although TRITC gave a more intense label (Table 1 and adjacent 1 to 4 nevertheless, it is easily cytotoxic, it easily penetrates when it is added to the culture medium freely. PFB-F therefore seems to be a better indicator.

Example 3: Examination of intracellular penetration in the presence of PFB-F (pentafluorobenzoylaminofluorescein) or TRITC (tetramethylrhodamine isothiocyanate) and in the presence or not in the presence of polyethyleneimine

The intracellular penetration of liposomes containing 5% soybean lecithin, 0.01% PFB-F or TRITC in the presence or not in the presence of 0.5% 25 kDa polyethyleneimine (PEI) prepared according to the protocol described in Example 1 was analyzed after 24 hours incubation with human fibroblasts cultured in a 24-well plate as described in Example 2.

The results obtained show that the addition of PEI stimulates the penetration of the PFB-F liposomes 13.7-fold, while the addition of PEI reduces the penetration of TRITC liposomes 16-fold (see photographs in the annex, 1 to 4 ).

The further used indicator is therefore PFB-F or pentafluorobenzyolaminofluorescein at 0.01% in the liposome.

Example 4: Investigation of the stability of the liposomes containing pentafluorobenzylaminofluorescein (PFB-F)

The stability of the liposomes was analyzed by examining the release of the fluorescent indicator into the DF culture medium by spectrofluorometry; this study was performed as a function of pH, ionic strength and the presence of a detergent.

Suitably, liposomes containing 5% soy lecithin, 0.01% PFB-F and 50 μM 25 kDa PEI were prepared. The liposomal solutions were adjusted to pH 6-7-8-9; the concentration of sodium chloride (NaCl) was adjusted to 50-100-150-200 mM and the concentration of sodium dodecyl sulfate (SDS) or Triton X-100 was adjusted to 0.1-0.5-1-3%. The results obtained are shown in Table 3. Table 3: Examination of release as a function of the presence of detergents, ionic strength and pH molecules concentrations Release: mean Release: standard deviation Triton X-100 3% 44027 177 1% 45800 2888 0.5% 40834 39 0.1% 19829 103 SDS 3% 62481 180 1% 71809 1558 0.5% 76676 200 0.1% 22990 108 NaCl 200 mM 68527 2040 150 mM 7622 36 100 mM 6314 113 50 mM 5680 62 pH 9 25265 411 8th 18816 226 7 8781 70 6 7640 55

The results appear to indicate that the liposomes containing 50 μM 25 kDa PEI began to destabilize from 200 mM NaCl, 0.5% Triton X-100 or SDS and above pH 7.

Example 5: Yield of encapsulation of the fluorescent indicator PFB-B as a function of the concentration of polyethyleneimine

Liposomes were prepared according to the procedure described in Example 5, with increasing concentrations of 25 kDa PH from 0 to 100 μM. After centrifugation at 1790 g for 10 minutes, the supernatants were quantified by spectrofluorometry. The yields of the encapsulation were calculated with reference to the initial concentration. The results are shown in Table 4. Table 4: Yield of encapsulation of PFB-F as a function of the concentration of PEI Concentration of PEI (μM) 0 1 10 50 100 Yield (%) 89.0 92.3 91.0 87.5 86.3

It should be noted that the yield of the encapsulation is not affected by the increase in the concentration of PEI.

Example 6: Development of Screening Conditions with Fluorescent Liposomes Containing Various Levels of Polyethyleneimine

Liposomes were prepared according to the protocol described in Example 1, with various concentrations of polyethylenimine (25 kDa PEI, neutralized with 6N hydrochloric acid), d. H. 0-5-10-50 μM and 0.01% PFB-F.

The quantification was determined by spectrofluorometry after incubation with fibroblasts cultured in a single layer in a 24-well plate for 2-4-6-24 and 48 hours, after three washes with phosphate buffer pH 7.4, against a blank of untreated fibroblasts and after normalization against free PFB-F at the same concentration. The results are shown in Table 5. Table 5: Examination of the intracellular penetration of PFB-F as a function of time and the concentration of PEI (n = 4, for each condition) expressed in arbitrary fluorescence units Concentrations of PEI 0 5 μM 10 μM 50 .mu.M Time Average standard deviation Average standard deviation Average standard deviation Average standard deviation 2 hours. 6 0 49 4 67 7 116 6 4 hours 7 3 63 5 89 9 159 10 6 hours 6 0 65 10 94 13 203 12 24 hours 9 3 120 5 129 3 291 17 48 hours 6 1 160 18 171 13 306 6

The best results were obtained at a concentration of 50 μM PEI and a minimum incubation time with the fibroblasts of 24 hours. These conditions are maintained for screening for molecules that stimulate intracellular penetration. The results are statistically significant for the free control sample (p <0.05) for all times with effect from the 5 μM concentration.

Example 7: Analysis of Intracellular Penetration and Cytotoxicity of Fluorescent Liposomes Containing 50 μM Polyethyleneimine as a Function of Time

The experiment was carried out according to Example 6 for PEI concentrations of 0 to 100 μM. The quantification of the fluorescence was carried out according to Example 6. The stimulation of intracellular penetration was expressed as a stimulation factor with respect to liposome without PEI.

Cell viability was performed in a 96-well plate (250 μl liposomal solution per well) by a test which evaluates alkaline phosphatase activity on cell mats (test with paranitrophenyl phosphate, n = 6). The results of cell viability were expressed as a percentage of live cells relative to a PEI-free fluorescent liposome control. The determination of IL1 alpha (Kit Quantikine R & D System) was carried out in the culture medium collected 24 hours after incubation, in parallel with a determination of total proteins (Bradford, Sigma). The results are shown in Tables 6 and 7. Table 6: Factor of intracellular penetration of PFB-F and cytotoxicity as a function of time and concentration of PEI (n = 4, for each condition) Time 0 5 μM 10 μM 50 μM Viability of PEI 50 2 hours. 0.7 5.4 7.4 12.9 93.8% 4 hours 0.8 7.0 9.9 17.7 92.4% 6 hours 0.7 7.2 10.4 22.6 83.9% 24 hours 1.0 13.3 14.3 32.3 72.6% 48 hours 0.7 17.8 19.0 34.0 48.4% Table 7: Determination of IL1-alpha in pg / mg of the total protein 25 kDa PEI 0 10 μM 50 μM 100 μM Total proteins in μg / ml 132.8 136.2 119.5 79.2 IL1 in pg / ml 9.2 12.7 13.1 14.4 IL1 in pg / mg of proteins 69.3 93.2 109.6 181.8 stimulating factor 1 1.3 1.6 2.6

The results show that it is possible in this experiment to increase the penetration factor of the fluorescent indicator in the presence of PEI up to 34-fold. However, it was observed that at the concentration of 50 μM of 25 kDa PH with effect from 24 hours, a considerable occurrence of cytotoxicity was observed, with more than half of the cells dead at 48 hours. As for the synthesis of IL1-alpha at the concentration of 50 μM, the stimulation is 1.6-fold, and at the concentration of 100 μM it is 2.6-fold. This molecule produces a significant inflammatory stress.

Example 8: Screening for molecules that stimulate the intracellular penetration of a fluorescent indicator incorporated into a liposome

Liposomes containing 5% soybean lecithin, 0.01% PFB-F, and 1-0.1-0.01% of the molecule to be tested were neutralized to pH 7 with hydrochloric acid, if necessary in Trizma dilution buffer, as in Example 5, prepared.

55 molecules were initially tested in triplicate, compared to the 50 μM 25 kDa PEI. Intracellular penetration was quantified 24 hours after incubation with normal human fibroblasts cultured in 24-well plates as described in Example 2. Cell viability was determined in a 96-well plate with 200 μl liposomal suspension. The results obtained for the concentration of 1% are given in Table 8 (Annex 2). From the screening performed, 15 molecules were selected because they were able to stimulate the intracellular penetration of the fluorescent indicator by more than 10%.

The cell viability was different (from 37 to 99%) at a concentration of 1%. Visualization of the fluorescent indicator confirmed the penetration for the molecules that stimulated more than 32% penetration. These molecules were preferably used further.

Example 9: Influence of the structure of the molecules on the stimulation of intracellular penetration

The preparation of the liposomes with primary fatty monoamines having an alkyl chain length of 3 to 18 carbons (carbon atoms) and the quantification of the intracellular penetration of the fluorescent marker were performed as in the example described above. The results are given in Table 9. Table 9: Stimulation of intracellular penetration as a function of carbon chain length Number of carbons (carbon atoms) Primary monoamine with a single alkyl fatty chain stimulating factor 25 kDa PEI control 34.7 C3 0.9 C4 1.4 C6 0.2 C7 3.7 C10 39.6 C11 95.3 C12 47.1 C13 44.5 C14 11.6 C15 14.9 C18 19.6

The incorporation of fatty amines with alipathic chains of different sizes into the membranes of the liposomes clearly shows the influence of the length of the carbon chains on the stimulatory activity of the intracellular penetration of PFB-F encapsulated in liposomes with 5% lecithin. There is a rough classification of fatty acids as a function of chain length. Thereafter, short chains (less than 8 carbons), middle chains (from 10 to 18 carbons) and long chains (more than 18 carbons) are distinguished. By exploiting this classification for the primary fatty amines tested in the context of this study, it appears that the middle chains are those that dissipate their ability to stimulate intracellular penetration of the liposomes to the liposomes.

Example 10: Influence of steric hindrance on intracellular penetration

To best determine the properties of the molecules with a stimulating effect on the intracellular penetration of a fluorescent indicator encapsulated in 5% lecithin liposomes, we have observed the influence of steric hindrance on the potential stimulatory activity of these molecules. We investigated the intracellular penetration of PFB-F encapsulated in liposomes in the presence of primary fatty monoamine and secondary fatty monoamine. The preparation of the liposomes with primary or secondary fatty amines and the quantification of the intracellular penetration of the fluorescent marker were performed as described in Example 8. The results are shown in Table 10. The diamines did not allow for better intracellular penetration of the marker. Table 10: Stimulation of intracellular penetration as a function of the number of carbon chains Number of alkyl chains of the amine (chain length) stimulating factor 25 kDa PEI 34.7 1 (C4) 1.7 2 (C4) 0.7 1 (C10) 39.6 2 (C10) 1.2

Example 11: Influence of steric hindrance on intracellular penetration

By operating as described in particular in Example 9 or 10, it has been shown that the length of the carbon chain is not the only factor affecting the stimulation of the intracellular penetration of the liposomal content.

R_Y stellt das hydrolysierte und hydroxypropylierte Sojaprotein dar
R_A stellt das hydrolysierte und hydroxypropylierte Weizenprotein darIn fact, in a comparison of the chemical structures shown in the following figure and encapsulated in liposomes, a group R specificity interfering with the C ₁₂ carbon chain was shown.

R _Y represents the hydrolyzed soy protein and hydroxypropylated
R _A represents the hydrolyzed and hydroxypropylated wheat protein

The quaternized molecules A or Y are products that are well known to those skilled in the art. The steric hindrance of the incorporated quaternized molecules (conventional molecules) plays a role in stimulating intracellular penetration of the liposomes with lecithin.

For example, the molecules of the following classes (Table 11) can be used at 1% to stimulate intracellular penetration to varying degrees up to + 39%. Other quaternized hydrolysates of molecules extracted from almonds, peas, potatoes or algae can also be used. Examples of conventionally available quaternized molecules Chemical name Examples of potential providers wheat Cocodimonium, steardimonium, hydroxypropyltriomonium or lauryldimonium hydrolyzed wheat protein. Wheat germ Amidopropalkoniumchlorid Croda soy Lauryldimonium hydroxypropyl-hydrolysed soy protein, cocodimonium hydroxypropyl-hydrolyzed soy protein, hydroxypropyltrimethylammonium chloride-hydrolyzed soy protein soy dihydroxypropyldimonium glucoside Croda RITA Corporation Keratin (non-invention) Hydroxypropyltrimonium hydrolyzed keratin RITA Corporation Casein (not according to the invention) Hydroxypropyltrimonium hydrolyzed casein RITA Corporation Collagen (non-invention) Hydroxypropyltrimonium-hydrolyzed collagen, Lauryldimoniumhydroxypropyl-hydrolyzed collagen RITA Corporation Cognis Silk (non-invention) Hydroxypropyltrimonium-hydrolyzed silk RITA Corporation rice Cocodimonium hydroxypropyl hydrolyzed rice protein SOCHIBO Guar (tufted bean) (non-inventive) Hydroxypropylguarhydroxypropyltrimonium chloride Hydroxypropylguar Hydroxypropyltrimonium chloride, guar hydroxypropyltrimonium Meyhall Rhodia Honey (non-inventive) Hydroxypropyltrimoniumhonig ARCH Cellulose (not according to the invention) Polyquaternium 4 and 10 Polyquaternium 39 Quimasso

Example 12 Optimization of Functionalizing Molecule Concentration (Stimulating Intracellular Penetration)

The stimulation of the intracellular penetration of the fluorescent indicator was optimized by testing different concentrations of functionalizing molecules.

Suitably, liposomes were formed with 5% soybean lecithin, 0.01% PFB-F and 0.5 to 2.5% of the quaternized soy solution in Trizma compound buffer. Intracellular penetration and cytotoxicity were evaluated as described in Example 2. The results obtained are shown in Table 12. Table 12: Examination of the concentration of a molecule that stimulates both intracellular penetration and stimulation factor and cell viability Quaternised soy in% 0.5 1 1.5 2 2.5 stimulating factor 0.6 1.6 38.9 97.2 99.9 Cell viability (%) 92.4 97.9 87.9 75.4 77.3

The sought-after intracellular penetration can thus be adjusted as a function of the concentration of the functionalizing agent and the maximum of the tolerated cytotoxicity.

Example 13: Inflammatory Effect Compared Between Polyethyleneimine and the Selected Quaternized Soy

The stimulation of the synthesis of interleukin 1 alpha is compared between liposomes functionalized by 50 μM PEI and by various concentrations in quaternized soybean solution.

Suitably, liposomes were formed with 5% soybean lecithin, 0.01% PFB-F, and 0.5 to 2.5% of the solution of quaternized soy or 50 μM PEI in Trizma dilution buffer. The IL-1 alpha content was evaluated with a kit (Quantikine R & D System) as described in Example 7. A test with paranitrophenyl phosphate (PNPP) was performed in parallel to evaluate the number of cells per well. The results of the IL1 alpha content were compared with the obtained optical density of PNPP. The results are shown in Table 13. Table 13: Effect of functionalizing molecules (PEI and quaternized soy) on the synthesis of interleukin 1 alpha Concentration of the functionalizing molecule 50 μM PEI 0.5% soy 1% soy 1.5% soy 2% soy PNPP 72.3 91.8 91.6 87.5 88.4 IL1 alpha 350 137.5 137.5 212.5 187.5 IL1 alpha / PNPP 484.1 149.8 150.1 242.9 212.1

The results obtained show that the solution of quaternized soy, which was previously selected, induces cytotoxicity and inflammatory stress, which is very limited compared to the reference molecule PEI.

Example 14: Apoptotic effect, compared between the polyethyleneimine and the selected quaternized soy

The stimulation of the synthesis of interleukin 1 alpha was compared between liposomes functionalized by 50 μM PEI and by various concentrations in solution of quaternized soy.

Suitably, liposomes were formed with 5% soybean lecithin, 0.01% PFB-F, and 0.5 to 2.5% of the solution of quaternized soy or 50 μM PEI in Trizma dilution buffer. The content of caspase 1 was evaluated with a kit (Caspase-1 Colorimetric Assay R & D System). A paranitrophenyl phosphate (PNPP) assay was performed in parallel to evaluate the number of cells per well. The results of caspase 1 content were compared with the obtained optical density of PNPP. The results obtained are shown in Table 14. Table 14: Effect of functionalizing molecules on caspase-1 content Concentration of the functionalizing molecule 50 μM PEI 0.5% soy 1% soy 1.5% soy 2% soy PNPP 59.8 91.8 91.6 87.5 88.5 Caspase-1 642.8 76 87.7 78.3 117.5 Caspase-1 / PNPP 1,074.9 82.8 95.7 89.5 132.8

The results obtained show that the solution of the previously selected quaternized soy induces cytotoxicity and apoptotic stress, which is excessively limited compared to the reference molecule PEI.

Example 15: Observation of the Structure of the Liposomal Composition of Example 14 by Transmissioris Electron Microscopy

The liposomes prepared with 2% quaternized soy solution without addition of a drug and a fluorescent indicator were observed by transmission electron microscopy. Negative staining of the liposomes was performed using heavy metal salts which disrupt the bilayers of multilamellar vesicles. The observation was carried out with a Philips CM120 transmission electron microscope and a magnification of 30 to 60,000.

The observations revealed the presence of concentric lipid layers characteristic of multilamellar structures. The average size of the observed liposomes varied from 150 to 250 nm.

Example 16: Stimulation of Anti-Radical Protection by Using a Quaternized Soy Solution of Example 11.

Liposomes were prepared with 5% soy lecithin, 0.2% natural vitamin E and 2% quaternized soybean solution from example 11 (molecule Y).

Conveniently, 0.4 g of natural vitamin E was added to 10 g of soy lecithin and 10 ml of 96% ethanol. The solution was evaporated for one night with magnetic stirring at room temperature and in the dark. After evaporation of the ethanol, 100 ml of deionized water was added. The mixture was stirred until complete dissolution.

The liposomes were then prepared by adding 5 ml of the lecithin vitamin E solution, 190 μl (ie 2%) quaternized soybean solution and 4.80 ml of 55 mM Trizma Dilution Buffer - 27 ml. NaCl adjusted to pH 5 and stored magnetic stirring in the dark for 30 minutes. The solution then became homogenized at maximum speed for 10 minutes to form the modified liposomes. The same solution was prepared without the homogenization and was a free vitamin E control.

The natural vitamin E-based liposomes were prepared in the presence of 10 μM 25 kDa PEI to prevent cytotoxicity and also without functionalizing agent.

A

= bestrahlt/nicht bestrahlt (1/NI)

B

= (I/NI Liposom) (I/NI Kontrollprobe)

C

= liposomiertes Vitamin E/freies Vitamin E

Normal human fibroblasts were seeded in 24-well plates and cultured to confluency. After rinsing three times with phosphate buffer with calcium and magnesium, pH 7.4 (In Vitrogen), the various solutions, diluted to half (ie final concentration of vitamin E of 0.1%), were incubated in culture medium for at least 2 hours. Wells were incubated with culture medium alone, which served as a control sample in the following procedure. After rinsing three times in phosphate buffer with calcium and magnesium, pH 7.4, to remove the liposomes and the free vitamin E, the cell mats were incubated in the presence of dihydrorhodamine 123 (Molecular Probes) in an amount of 200 μl / well of a solution as described 150 μl of a 1 mM solution dissolved in 15 ml HBSS buffer was incubated. The plate was read for fluorescence at 490 to 530 nm and then exposed to UVB at 912 nm at 0.8 J / cm ² and read again for fluorescence at the same wavelength. The results are expressed in ratios:

A

= irradiated / not irradiated (1 / NI)

B

= (I / NI liposome) (I / NI control)

C

= liposomed vitamin E / vitamin E free

The fluorescent sample used allowed the evaluation of the presence of intracellular reactive oxygen intermediates as it passively diffuses through the cell membranes where it can then be oxidized to cationic rhodamine. For example, the fluorescent sample reacted positively with hydrogen peroxide and peroxynitrite. The results obtained are given in Table 15. Table 15: Anti-radical protection by the thus modified liposomes I NI A = 1 / NI B = liposome / control C = liposome / free Product of the invention 118990 58786 2.01 79.3% + 23% Non-functionalized liposome 114907 52306 2.31 91.1% + 11% Liposome PEI 105257 40148 2.82 111.4% -9%

The results indicate that the liposomes functionalized by the quaternized soy protein make it possible to achieve a more than 20% higher protective effect against radical stress compared to non-functionalized liposomes, whereas the liposomes functionalized with the reference molecule PEI did not show a protective effect. but create additional stress.

Example 17: Stimulation of depigmentation by the products of the invention

Liposomes were prepared according to the following protocol: 5% soy lecithin to which 2% quaternized soybean solution or 10 μM PEI (reference liposome) was added or not added (reference liposome). The active compounds have also been co-encapsulated: Phytolight ^® (Coletica, Lyons, France) without preservative at 0.05% (cocktail of botanicals), 0.05% arbutin and 1 mM catechin (Sigma). The molecules were freely applied, in 5% lecithin liposome, in 10 μM PH functionalized liposomes or 2% quaternized soy, on normal human melanocytes cultured in 24 well plates and pre-confluent (prior to pooling). The drugs which were liposomed or non-liposomed were incubated for 66 hours at 37 ° C under 5 CO ₂ in MMK2 medium (Sigma). After three rinses with. Phosphate buffer with calcium and magnesium (Invitrogen) and extraction in phosphate buffer containing 0.5% Triton X-100, the tyrosinase activity was quantified by determination in the presence of L-DOPA and MBTH (3-methyl-2-benzothiazolinone hydrazone). The formation of a MBTH-o-quinone compound was quantified kinetically by an absorbance value at 490 nm. The tyrosinase activity was expressed by the increase (rate) of the enzyme reaction. The results are shown in Table 16. Table 16: Depigmentation effect of liposomed or non-liposomed drugs (expressed in the rate of enzyme reaction) Free Liposome (reference liposome) PEI liposome (reference liposome) Soy liposome arbutin 0,004 0.001 0 0 Phytolight ^® 0.01 0,008 0,003 0.0016 catechin 0,007 0,007 0 0,003

The results show that as a function of the co-encapsulated drugs, functionalization allows an increase in the inhibition of tyrosinase activity in vitro on normal human melanocytes. The functionalization by the quaternized soy is more effective than the functionalization by the addition of PEI for the complex of plant extracts, is equivalent to arbutin and is slightly lower for catechin. In any case, the activity is at least 2.3-fold higher than with an unfunctionalized liposome.

Example 18: Method of reducing the size of the products of the invention

The size of the liposomes can be reduced by using a high-pressure homogenizer (with a working pressure of more than 1,000 bar, preferably more than 2,000 bar, more preferably more than 3,000 bar). At 3,000 bar, liposomes of about 50 nm can be obtained with this device.

The size of the liposomes was analyzed by means of a laser particle size analyzer (Granulometer) (Beckman Coulter, N4 plus, Submicron Particle Size Analyzer) and by transmission electron microscopy as described in Example 15.

Example 19: Representation of the penetration of material carried by the products of the invention after transcutaneous permeation: Examples with a fluorescent indicator, vitamin E or genetic material

Penetration of a fluorescent molecule (0.01% PFP-F), free or incorporated into a liposome as described in Example 12, which included or did not comprise 2% quaternized soybean solution, selected from Example 11, became more human by transcutaneous permeation Skin quantified. The diffusion kinetics were quantified spectrofluorometrically from 3 to 24 hours. The release was quantified after another 24 hours. The storage was also evaluated last. The number of samples tested was 5 per test condition. The results obtained in Table 17 show that the use of liposomes made in the presence of quaternized soy significantly stimulates the diffusion, release and incorporation of the fluorescent indicator as compared to the non-vectored form or form, which was vectorized without quaternized substance. Table 17: Free liposome Quaternized liposome diffusion 0.57 ± 0.03 1.17 ± 0.05 1.76 ± 0.13 Release 24 hours 0.691 ± 0.06 1,398 ± 0.07 1.77 ± 0.21 warehousing 0.81 ± 0.1 1.27 ± 0.17 2.61 ± 0.33

The penetration of the vitamin E molecule (0.5%), which was free or incorporated into a liposome as described in Example 12, and the 2% quaternized soybean solution selected from Example 11, included or not included, was determined by transcutaneous Permeation quantified on human skin. The diffusion kinetics were quantified by high performance liquid chromatography for 5 to 24 hours. The release was quantified after another 24 hours. The storage was also evaluated last. The results obtained also show that the use of liposomes made in the presence of quaternized soy significantly stimulates the diffusion, release and incorporation of vitamin E as compared to the non-vectored form or the form without quaternization Substance was vectorized.

The same experiment of transcutaneous penetration was performed with incorporation (or not) of fluorescent genetic material (200 mM) into a liposome as described in Example 12 in the presence of a 2% soy solution. The results obtained by observing transversal sections of the skin by optical epifluorescence microscopy show that the use of liposomes made in the presence of quaternized soy allows diffusion of the genetic material down to the deep dermis.

Sequence of the fluorescent probe from duplex - ("duplexed") elastin 19-20:
Sense: 5 '- (FITC) AGCUGCUAAGGCUGGCGCUTT-3'
Antisense: 5'-AGCGCCAGCCUUAGCAGCUTT-3 '

Example 20: Use of the products of the invention in cosmetic or pharmaceutical formulations of the oil-in-water emulsion type

Formulation 20a: A water qsp 100 butylene 2 glycerin 3 Natriumdihydroxycetylphosphat, 2 Isopropylhydroxycetylether B Glycolstearate SE 14 Triisononaoin 5 octyl cocoate 6 C Butylene glycol, methylene paraben, 2 ethyl paraben, Propylparaben, pH adjusted to 5.5 D Products of the invention 0.01-10% Formulation 20b: A water qsp 100 butylene 2 glycerin 3 Polyacrylamide, isoparaffin, 2.8 Laureth-7 B butylene glycol, 2 methylparaben, Ethyl paraben, propyl paraben; 2 phenoxyethanol, methylparaben, Propylparaben, Butylparaben, Ethylparaben 0.5 butylene D Products of the invention 0.01-10% Formulation 20c: A Carbomer 0.50 propylene glycol 3 glycerin 5 water qsp 100 B octyl cocoate 5 bisabolol 0.30 dimethicone 0.30 C sodium hydroxide 1.60 D phenoxyethanol, 0.50 methylparaben, Propylparaben, Butylparaben, Ethylparaben e fragrances 0.30 F Products of the invention 0.01-10%

Example 21: Use of the products of the invention in a water-in-oil type formulation

A PEG 30 dipolyhydroxystearate 3 capric triglycerides 3 cetearyl 4 dibutyl 3 Grapeseed oil 1.5 jojoba oil 1.5 phenoxyethanol, 0.5 methylparaben, Propylparaben, Butylparaben, Ethylparaben B glycerin 3 butylene 3 magnesium sulfate 0.5 EDTA 0.05 water qsp 100 C cyclomethicone 1 dimethicone 1 D fragrances 0.3 e Products of the invention 0.01-10%

Example 22: Use of the products of the invention in a formulation of a triple emulsion type (triple emulsion type)

First emulsion W1 / O A PEG 30 dipolyhydroxystearate 4 capric triglycerides 7.5 Isohexadecan 15 PPG-15 stearyl ether 7.5 ("PPG-15 stearul ether") B water 65.3 C phenoxyethanol, 0.7 methylparaben, Propylparaben, Butylparaben, Ethylparaben Second emulsion W1 / O / W2 A First emulsion 60 B Poloxamer 407 2 phenoxyethanol, 0.3 methylparaben, Propylparaben, 2-bromo-2-nitro propane-1,3-diol water qsp 100 C Carbomer 15 D triethanolamine pH 6.0-6.5 e Products of the invention 0.01-10%

Example 23: Use of the products of the invention in a lipstick-type formulation and other anhydrous products

A mineral wax 17.0 isostearyl isostearate 31.5 propylene glycol dipelargonate 2.6 propylene glycol isostearate 1.7 PEG 8 beeswax 3.0 Hydrogenated palm kernel oil 3.4 glycerides hydrogenated palm glycerides lanolin 3.4 sesame oil 1.7 cetyl lactate 1.7 Mineral oil, lanolin alcohol 3.0 B Castor oil qsp 100 Titanium dioxide 3.9 Cl 15850: 1 0.616 Cl 45410: 1 0.256 Cl 19140: 1 0.048 Cl 77491 2.048 C Products of the invention 0.01-5%

Example 24: Use of the products of the invention in a formulation of aqueous gels (eyeliners, amincissants, slimmers, etc.)

A water qsp 100 Carbomer 0.5 butylene 15 Phenoxyethanol, methylparaben, 0.5 Propylparaben, Butylparaben, Ethylparaben B Products of the invention 0.01-10%

Example 25: Preparation of pharmaceutical formulations containing the product of the invention

Formulation 25a: preparation of tablets A carrier in g per tablet lactose 0.359 sucrose 0.240 B Product of the invention 0.001-0.1 Formulation 25b: Preparation of an ointment A carrier Low density polyethylene 5.5 Liquid paraffin qsp 100 B Product of the invention 0.001-0.1 Formulation 25c: Preparation of an injectable formulation A carrier Isotonic saline 5 ml B Product of the invention 0.001-0.1 g

Phase A and Phase B are packed in separate vials and mixed before use.

Example 26: Evaluation of Cosmetic Acceptance of the Products of the Invention

Toxicological tests were carried out with the compounds obtained according to Example 15 (without incorporation of active substance), by skin evaluation and ocular evaluation in the rabbit, by the study of the absence of abnormal toxicity in single oral administration to the rat and by the investigation of sensitizing power in guinea pigs.

Evaluation of the initial irritation of the skin in rabbits:

The preparations described above were applied without dilution at a dose of 0.5 ml to the skin of three rabbits, according to the procedure recommended by the OECD guideline regarding the study "the acute irritant / corrosive effect on the skin" becomes.

The products were classified according to the criteria defined in the decision of 1/2/1982, published in the Official Journal of the French Republic (the "JORF") of 21/02/82.

The results of these tests led to the conclusion that the preparation containing the compound obtained according to Example 12 was classified as non-irritating to the skin.

Evaluation of ocular irritation in rabbits:

The preparations described above were pure and instilled into the eyes of three rabbits in a single administration of 0.1 ml according to the procedure described by the OECD Guideline No. 405 of 24 February 1987 relating to the study "the acute irritant / caustic effect on the eyes "was proposed.

The results of this test lead to the conclusion that the preparations can not be considered as irritating to the eyes.

Test for absence of abnormal toxicity by single oral administration to the rat:

The preparations described were administered in a single oral administration at the dose of 5 g / kg body weight to 5 male rats and to 5 female rats, in accordance with a protocol inspired by the OECD Guideline No 401 of 24 February 1987 cosmetic products was adjusted.

It was found that LD0 and LD50 are greater than 5000 mg / kg. The tested preparations are therefore not classified among the preparations that are dangerous when ingested.

Evaluation of the Skin Sensitizing Potential in Guinea Pigs:

The formulations described were subjected to a maximization test described by Magnusson and Kligmann, a protocol which complies with OECD Guideline No. 406. The preparations are classified as non-sensitizing on contact with the skin.

Evaluation of the mutagenicity potential:

The protocol complies with the OECD Guideline 471 (Directive 92/69 / EC).

The mutagenesis assays were performed with "Salmonella typhimurium" and with "Escherichia coli" according to the method of Ames et al. (Mutation Research, 1975, 31, 347-364). Five strains were exposed to the product of the invention in minimal medium, with or without exogenous systems of metabolism activation (to distinguish pro-mutagens and direct mutagens). After incubation, the mutant colonies were counted and compared to the number of colonies that spontaneously mutated under controls.

The product of the invention has no mutagenic activity within the meaning of Directive 92/69 / EC).

Evaluation of sensitization potential in healthy volunteers (subjects):

Evaluation of the allergenic potential of the product of the invention on a panel (a group) of volunteers (subjects). The protocol is in accordance with the method of Marzulli and Maibach (Contact Dermatitis, 1976, 2, 1-17), which includes an induction phase and a triggering test ("épreuve déclenchante"). This test was conducted with a panel (panel) of 100 healthy volunteers (subjects) of female and / or male sex, aged 18 to 65 years, of any skin type.

A patch occlusive patch containing the product of the invention diluted to 20% was applied to the shoulder area of each of the volunteers (subjects). The patches were left in direct contact with the skin for 24 hours and were applied repeatedly every two days for 3 weeks for a total of 9 applications. After removal of each patch, the clinical signs of irritation and skin sensitization were evaluated = induction phase.

After a period of 2 weeks, other patches containing the product of the invention, diluted to 20%, were applied to the skin of the volunteers (subjects) and left in direct contact with the skin surface for 24 hours. Clinical signs of irritation and skin sensitization were evaluated 24, 48 and 72 hours after removal of the patch = "Challenge Phase".

None of the 100 volunteers (subjects) involved in this study showed clinical signs of irritation or skin sensitization, either during the induction phase or during the challenge phase.

Anhang 2: Ergebnisse des Screenings nach Molekülen, welche die Penetration stimulieren (Tabelle 8). INCI-Name Stimulierungsfaktor Lebensfähigkeit Visualisierung Polyethylenimin 25 kDa 34,7 74,1 + Cocodimoniumhydroxypropyl-hydrolysiertes Weizenprotein 4,3 86 - Cocodimoniumhydroxypropyl-hydrolisiertes Reisprotein 18 85 - Steadimoniumhydroxypropyl-hydrolysiertes Weizenprotein 4,6 86,4 - Lauryldimoniumhydroxypropyl-hydrolysiertes Weizenprotein 6,2 87,5 - Hydroxypropyltrimonium-hydrolysiertes Weizenprotein 1,3 91,7 - Hydroxypropyltrimoniumhonig 15 92 - Natriumlauroylhafer-Aminosäuren 0,8 76,6 - Natriumlauroylweizen-Aminosauren 1,1 46,1 - Cocoamin 32,7 79,7 + Lauryldimoniumhydroxypropyl hydrolysiertes Sojaprotein 38,8 87,9 + Chitosan 10,3 99 - Hydroxypropylguarhydroxy-propyltrimoniumchlorid 1,1 74,8 - Hydroxypropylguar –0,2 91,1 - Hydroxypropylguar –0,4 97,5 - Hydroxypropylguar-hydroxypropyltrimoniumchlorid 1,4 71,2 - Guarhydroxypropyltrimonium 0,6 53,3 - Meypro 1 91,6 - Polyquaternium 7 0,3 80,1 - Polyvinylpyrrolidon (1) 1,4 85,1 - Polyvinylcaprolactam 4,1 0 - Polyvinylpyrrolidon (2) 0,7 26,6 - Polyvinylpyrrolidon (3) 0,7 71 - Polyquaternium 16 (1) 4,6 83,5 - Polyquaternium 11(1) 1,5 49,7 - Cocotrimoniummethosulfat 0,1 55 - Polyquaternium 16 (2) 8,4 33,8 - Polyquaternium 16 (3) 1 38,8 - Polyquaternium 16 (4) 12,5 67,1 - Polyquaternium 16 (5) 0,2 85,3 - Polyquaternium 44 0,8 59,7 - Cocoalkoniumchlorid 5,6 36,3 - Cocotrimoniumchlorid –0,2 65,6 - Tetrahydroxypropylethylendiamin 1,9 95,3 - Stearamidopropylcetearyldimoniumtosylat und PG 13,1 93,2 - Quaternium 70 und PG –0,2 63,9 - Quaternium 26 30,9 59,2 - Quaternium 22 2 98,9 - Polyquaternium 28 0,6 79,3 - Polyquaternium 11 (2) 2,9 50,7 - Polyquaternium 11 (3) 2,9 77,1 - Polyquaternium 2 4,2 23,6 - Stearalkoniumchlorid 17,6 37,4 - Propylamin C3 0,9 103,3 - n-Butylamin C4 1,4 106,2 - Dibutylamin 2 C4 0,7 75,7 - Hexylamin C6 0,2 82,1 - Heptylamin C7 3,7 39,4 - Dioctylamin 2 C8 1 34,7 - Decylamin C10 39,6 68,5 + Didecylamin 2 C10 1,2 28,3 - Undecylamin C11 95,3 84 + Dodecylamin C12 47,1 57 + Tridecylamin C13 44,5 62,6 + Tetradecylamin C14 11,6 54,8 - Pentadecylamin C15 14,9 73 - Octadecylamin C18 19,6 61,2 - Oleylamin C18:1 1,9 87,1 - ↑ Anhang 2, Tabelle 8 (Fortsetzung) Under the experimental conditions, the product of the invention, diluted to 20%, has no allergenic potential.

Appendix 2: Screening results for molecules that stimulate penetration (Table 8). INCI name stimulating factor viability visualization Polyethyleneimine 25 kDa 34.7 74.1 + Cocodimonium hydroxypropyl-hydrolyzed wheat protein 4.3 86 - Cocodimonium hydroxypropyl hydrolyzed rice protein 18 85 - Steadimony hydroxypropyl-hydrolyzed wheat protein 4.6 86.4 - Lauryldimonium hydroxypropyl-hydrolyzed wheat protein 6.2 87.5 - Hydroxypropyltrimonium-hydrolyzed wheat protein 1.3 91.7 - Hydroxypropyltrimoniumhonig 15 92 - Natriumlauroylhafer-amino acids 0.8 76.6 - Natriumlauroylweizen-amino acids 1.1 46.1 - cocoamine 32.7 79.7 + Lauryldimonium hydroxypropyl hydrolyzed soy protein 38.8 87.9 + chitosan 10.3 99 - Hydroxypropylguarhydroxy-propyltrimoniumchlorid 1.1 74.8 - hydroxypropyl -0.2 91.1 - hydroxypropyl -0.4 97.5 - Hydroxypropyl hydroxypropyltrimonium 1.4 71.2 - Guarhydroxypropyltrimonium 0.6 53.3 - Meypro 1 91.6 - Polyquaternium 7 0.3 80.1 - Polyvinylpyrrolidone (1) 1.4 85.1 - polyvinyl caprolactam 4.1 0 - Polyvinylpyrrolidone (2) 0.7 26.6 - Polyvinylpyrrolidone (3) 0.7 71 - Polyquaternium 16 (1) 4.6 83.5 - Polyquaternium 11 (1) 1.5 49.7 - Cocotrimoniummethosulfat 0.1 55 - Polyquaternium 16 (2) 8.4 33.8 - Polyquaternium 16 (3) 1 38.8 - Polyquaternium 16 (4) 12.5 67.1 - Polyquaternium 16 (5) 0.2 85.3 - Polyquaternium 44 0.8 59.7 - Cocoalkoniumchlorid 5.6 36.3 - Cocotrimoniumchlorid -0.2 65.6 - tetrahydroxypropylethylene 1.9 95.3 - Stearamidopropyl cetearyldimonium tosylate and PG 13.1 93.2 - Quaternium 70 and PG -0.2 63.9 - Quaternium 26 30.9 59.2 - Quaternium 22 2 98.9 - Polyquaternium 28 0.6 79.3 - Polyquaternium 11 (2) 2.9 50.7 - Polyquaternium 11 (3) 2.9 77.1 - Polyquaternium 2 4.2 23.6 - stearalkonium 17.6 37.4 - Propylamine C3 0.9 103.3 - n-butylamine C4 1.4 106.2 - Dibutylamine 2 C4 0.7 75.7 - Hexylamine C6 0.2 82.1 - Heptylamine C7 3.7 39.4 - Dioctylamine 2 C8 1 34.7 - Decylamine C10 39.6 68.5 + Didecylamine 2 C10 1.2 28.3 - Undecylamine C11 95.3 84 + Dodecylamine C12 47.1 57 + Tridecylamine C13 44.5 62.6 + Tetradecylamine C14 11.6 54.8 - Pentadecylamine C15 14.9 73 - Octadecylamine C18 19.6 61.2 - Oleylamine C18: 1 1.9 87.1 - ↑ Appendix 2, Table 8 (continued)

Claims (32)

Use of liposomes, characterized in that they comprise, at least in part, in their structure a substance or mixture of substances capable of, or the intracellular penetration of, at least one active substance present in or from the liposomes is to stimulate and which is selected from the group consisting of: a) a primary, secondary, tertiary or quaternary fatty monoamine comprising a single C10 to C13 fatty chain; or b) a solution of quaternized plant proteins of the formula RN (R ₁ R ₂ R ₃ ), where R symbolizes the vegetable protein molecule; R ₁ and R _{2 are} independently a C1-C6 hydrocarbon group, and R ₃ is an alkyl radical having 10 to 18 carbon atoms; as a cosmetic agent that stimulates the intracellular penetration of at least one drug to produce a topical cosmetic composition; or for the preparation of a topical pharmaceutical composition. Use according to claim 1, characterized in that the quaternized plant protein b) is selected from the group consisting of quaternized wheat proteins, quaternized rice protein, quaternized soy protein. Use according to claim 1, characterized in that the primary monoamine a) is selected from the group consisting of decylamine, undecylamine, dodecylamine, tridecylamine, or a mixture of primary amines having a hydrocarbon chain of C10 to C13. Use according to any one of the preceding claims, characterized in that the liposomes comprise at least one lecithin in the lipid phase extracted from a natural source selected from the group consisting of soya, rapeseed, sunflower, lupine, peanut, sesame, pumpkin, bran oil , large-sized glue dotter, marigold, linen (flax) and hemp, individually or in a mixture. Use according to any one of the preceding claims, characterized in that the liposomes in the lipid phase comprise cholesterol or cholesteryl hemisuccinate as a substance which stiffens the membranes. Use according to one of the preceding claims, characterized in that the liposomes in the lipid phase comprise at least one surfactant ("surfactant agent") or a surfactant as a substance which liquefies the membrane of the liposomes. Use according to any one of the preceding claims, characterized in that they contain a fluorescent substance or a fluorescent component which is substantially inert with respect to intracellular penetration, comprising approximately 0.01% by weight of 5-pentafluorobenzoylamine fluorescein, of the composition containing liposomes. Use according to any one of the preceding claims, characterized in that the concentration of the substance (s) which stimulate / stimulate intracellular penetration is from 0.05% to 25% by weight of a composition containing the liposomes. Use according to any one of the preceding claims, characterized in that the concentration of the substance (s) which stimulate / stimulate intracellular penetration is from 0.5% to 2.5% by weight of a composition containing the liposomes , Use according to any one of the preceding claims, characterized in that the fatty monoamine is a quaternized primary monoamine comprising a single fatty carbon chain of C10 to C13. Use according to any one of the preceding claims, characterized in that the quaternized vegetable protein is hydrolyzed or not, hydroxyalkylated or not. Use according to one of the preceding claims, characterized in that the quaternized vegetable protein is hydroxypropylated or not. Use according to any one of the preceding claims, characterized in that R ₁ and R _{2 of} the quaternized vegetable protein are methyl or ethyl. Use according to any one of the preceding claims, characterized in that R _{3 of} the quaternized vegetable protein has 12 carbon atoms (lauryl). Use according to any one of the preceding claims, characterized in that the quaternized vegetable protein of the formula RN (R ₁ R ₂ R ₃ ) is lauryldimonium hydroxypropyl-hydrolyzed soy protein or cocodimonium hydroxypropyl-hydrolysed soy protein. Use according to any one of the preceding claims, characterized in that the active substance present in or carried by the liposomes is selected from the group consisting of an antiradical-forming substance such as vitamin E, a flavonoid, a carotenoid, vitamin C; a depigmenting substance such as catechin, hydroquinone, arbutin, phytic acid, ellagic acid, vitamin C; a degrading (slimming) substance such as at least one xanthine; a skin or hair pigmenting substance such as tyrosine, tryptophan, phenylalanine, a coleus extract. Liposomes, characterized in that they at least partially in their structure a solution of quaternized plant proteins of the formula of the type RN (R ₁ R ₂ R ₃ ), wherein R symbolizes the plant protein molecule; R ₁ and R _{2 are} independently a C ₁ -C 6 hydrocarbon group and R _{3 is} an alkyl radical having from 10 to 18 carbon atoms; which stimulates the intracellular penetration of at least one drug present in or carried by the liposomes. Liposomes according to claim 17, characterized in that the quaternized vegetable protein is selected from the group consisting of quaternized wheat proteins, quaternized rice protein and quaternized soy protein. Liposomes according to claim 17 or 18, characterized in that the quaternized vegetable protein is hydrolysed or not, is hydroxyalkylated or not. Liposomes according to one of claims 17 to 19, characterized in that the quaternized vegetable protein is hydroxypropylated or not. Liposomes according to any one of claims 17 to 20, characterized in that R ₁ and R _{2 of} the quaternized vegetable protein are methyl or ethyl. Liposomes according to any one of claims 17 to 21, characterized in that R _{3 of} the quaternized vegetable protein has 12 carbon atoms (lauryl). Liposomes according to any one of claims 17 to 22, characterized in that the quaternized vegetable protein of formula RN (R ₁ R ₂ R ₃ ) is lauryldimonium hydroxypropyl-hydrolyzed soy protein or cocodimonium hydroxypropyl-hydrolysed soy protein. Cosmetic or dermocosmetic composition, characterized in that it comprises liposomes according to any one of claims 17 to 23 in a mixture with a cosmetically acceptable carrier. Pharmaceutical or dermopharmaceutical composition characterized in that it comprises liposomes according to any one of claims 17 to 23 in a mixture with a cosmetically acceptable carrier. Composition according to Claim 24 or 25, characterized in that the concentration of the substance which stimulates intracellular penetration is from 0.05% to 25% by weight of the final composition, preferably from 0.5% to 2.5% by weight -% of the final composition. A method of screening for at least one substance selected from a) a primary, secondary, tertiary or quaternary fatty monoamine comprising a single C10 to C13 fatty chain; or b) a solution of quaternized plant proteins of the formula RN (R ₁ R ₂ R ₃ ), where R symbolizes the vegetable protein molecule; R ₁ and R _{2 are} independently a C ₁ -C 6 hydrocarbon group and R _{3 is} an alkyl radical having from 10 to 18 carbon atoms; which is potentially capable of stimulating the intracellular penetration of a substance or drug comprising the liposomes according to claims 17-23 and liposomes which comprise at least partly in their structure a substance or a mixture of substances which are selected from the group consisting of a primary, secondary, tertiary or quaternary fatty monoamine comprising a single C10 to C13 fatty chain is carried; characterized in that it comprises: a) preparing a hydro-lipidic mixture which is capable of containing the liposomes according to claims 17-23 and liposomes which at least partly in their structure comprise a substance or a mixture of substances which are selected from the group consisting of a primary, secondary, tertiary or quaternary fatty monoamine comprising a single C10 to C13 fatty chain; b) adding a fluorescent compound, which is preferably substantially inert with respect to intracellular penetration, prior to the dispersion process d) to / in said hydrolipidic mixture; c) adding a test substance selected from the substance that is potentially active and that is capable of stimulating intracellular penetration and a reference substance in the hydro-lipidic mixture before or during the dispersion process d); d) dispersion process for the preparation of the liposomes from the hydro-lipic mixture as obtained either in step b) or in step c); e) incubation of the preparation with fibroblasts for 2 to 48 hours; f) detecting at least the intracellular penetration of the fluorescent compound by measuring intracellular fluorescence. A method according to claim 27, characterized in that the result of the intracellular penetration of the fluorescent compound is compared with the intracellular penetration achieved with a reference substance. A method according to claim 27 or 28, characterized in that the cytotoxicity and / or the induction of apoptosis of the substance which is potentially active is determined. A method according to any one of claims 27 to 29, characterized in that the fluorescent compound, which is substantially inert with respect to the intracellular penetration, is a fluorescent compound which does not spontaneously penetrate into fibroblasts in culture. A method according to any one of claims 27 to 30, characterized in that the fluorescent compound is or comprises pentafluorobenzoylaminofluorescein, which is used at a concentration of 0.01% by weight of the final composition containing the liposomes, which is used to evaluate intracellular penetration become. A method according to any one of claims 27 to 31, characterized in that the fluorescent compound is incorporated in the presence of polyethyleneimine, wherein the fluorescent compound and the polyethyleneimine are present in a non-cytotoxic concentration.

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